The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus and is associated with transformed cells, including several human cancer cell lines. Several small RNAs transcribed by RNA polymerase III and the hnRNP protein, the polypyrimidine tract binding (PTB) protein, have been localized in the PNC. Using a GFP-PTB fusion protein to follow the PNC in living cells, we showed that the PNC was a dynamic structure hat makes small movements at the periphery of and occasionally into the nucleolus over time. At the ultrastructural level, using anti-PTB antibodies and eosin fluorescence photooxidation, we observed that the PNCs are in direct contact with the surface of the nucleolus and occasionally extend into the nucleolus itself. The structure is highly irregular and no clear structural boundary was observed between the PNC and the remainder of the nucleoplasm. Stereo images from 1 5m thick sections viewed with IV EM revealed strand-like structures across the PNC. High resolution imaging of unstained PNCs was accomplished by using the GFP-PTB fluorescence to identify the locations of PNCs at the light microscopic level. Transfected cells were photographed, optimally fixed and processed for electron microscopic examination. The nuclear region corresponding to the PNCs appeared to be an electron-dense structure composed of thick, short strands measuring ~80-180 nm in daimeter. Each strand was surrounded by less electron-dense areas. Some of the strands were directly linked to the nucleolus and could be quite long in individual sections, up to 1 5m in length. We sought to determine if these thick strands are interconnected and if they form a higher order structure. To resolve the 3D organization of the PNC at high resolution, PNCs in serial thin sections of optimally fixed HeLa cell nuclei were examined by electron microscopy. Three-dimensional reconstructions revealed that the PNC is a highly interconnected and reticulated mesh associated with a portion of the nucleolar surface. However, there was considerable variability in the shape, size and the relationship of the PNC with the nucleolus. In some cells, the PNC was mostly positioned on the surface of the nucleolus, while in others, the PNC extended into the nucleolus like a plug. Occasionally, a portion of the nucleolus extended into the PNC. This variability may represent freeze frames of a dynamic subnuclear structure. These 3D reconstructions have shown that the PNC is structurally distinct from the nucleolus and forms a reticulated mesh associated with the nucleolar surface. Together, these findings suggest that the PNC may be a functional compartment involved in RNA metabolism in the nucleus of transformed cells. This work was published in the Journal of Cell Biology (Huang et al., J. Cell. Biol., 137: 965-974, 1997) and a second manuscript is in preparation.